Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.     

Chemical modification of lysine residues in lysozyme may dramatically influence its amyloid fibrillation

Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein–protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97.     

Propeptide-Mediated Inhibition of Cognate Gingipain Proteinases

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism’s cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K_i values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.     

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE*

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.     

Temporal and Contextual Consistency of Leadership in Homing Pigeon Flocks

Organized flight of homing pigeons (Columba livia) was previously shown to rely on simple leadership rules between flock mates, yet the stability of this social structuring over time and across different contexts remains unclear. We quantified the repeatability of leadership-based flock structures within a flight and across multiple flights conducted with the same animals. We compared two contexts of flock composition: flocks of birds of the same age and flight experience; and, flocks of birds of different ages and flight experience. All flocks displayed consistent leadership-based structures over time, showing that individuals have stable roles in the navigational decisions of the flock. However, flocks of balanced age and flight experience exhibited reduced leadership stability, indicating that these factors promote flock structuring. Our study empirically demonstrates that leadership and followership are consistent behaviours in homing pigeon flocks, but such consistency is affected by the heterogeneity of individual flight experiences and/or age. Similar evidence from other species suggests leadership as an important mechanism for coordinated motion in small groups of animals with strong social bonds.     

DNA methylation status of the methylenetetrahydrofolate reductase gene promoter in peripheral blood of end-stage renal disease patients

End-stage renal disease (ESRD) is one of the main causes of morbidity and mortality worldwide. DNA methylation is a major epigenetic modification of the genome that has the potential to silence gene expression. Methylenetetrahydrofolate reductase (MTHFR) gene inactivation was recognized as a predisposing factor of hyperhomocysteinemia in renal patients. The current study aimed to determine the methylation status within the MTHFR promoter region in DNA isolated from peripheral blood of ESRD patients and controls and the correlation of this methylation with the clinical and biochemical characteristics in ESRD patients. Ninety-six ESRD patients and 96 healthy ethnically, age and gender matched controls were included within the study. MTHFR promoter methylation was assessed using methylation specific polymerase chain reaction. The frequency of MTHFR methylation was significantly higher in ESRD patients than in controls (P = 0.003), additionally, MTHFR methylation was associated to a decrease in estimated glomerular filtration rate, serum high-density lipoprotein cholesterol level and an increase in both serum total cholesterol and low-density lipoprotein cholesterol levels. Data generated from this study suggest the possible involvement of MTHFR promoter methylation in the pathogenesis of ESRD and support a new dimension of MTHFR inactivation.     

Generalized motor programs for rapid bimanual tasks: a two-level multiplicative-rate model

A model is proposed for the timing of rapid bimanual movements. It combines (a) the notion of a generalized motor program (GMP) with invariant relative timing, (b) the two-level concept of timing control with a central level of control and a peripheral level where the observations are made, and (c) the hypothesis that a single GMP simultaneously controls both limbs. Our method is based on the analysis of temporal intervals measured among landmarks taken from the bimanual kinematic traces. We show that sets of tetrad ratios — each composed of two pairs of covariances among four temporal intervals in the actions — should be equal to 1.0 if the hypothesis is correct. In addition, we show that these tetrad ratios should deviate systematically from 1.0 under certain, biologically realizable violations of the model. Data from human subjects show that the results generally conform to the basic model. Simulations are used to illustrate other violations of the model and to explore characteristics of the sampling distribution of the tetrad ratios under the model.